Publications by authors named "Jeffrey S Karns"

45 Publications

Diversity of Extended-Spectrum Cephalosporin-Resistant in Feces from Calves and Cows on Pennsylvania Dairy Farms.

Foodborne Pathog Dis 2019 05 4;16(5):368-370. Epub 2019 Feb 4.

1 Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, USDA-ARS, Beltsville, Maryland.

The global incidence of human infections associated with extended-spectrum β-lactamase (ESBL)-producing is increasing. Dairy animals are reservoirs of ESBL-producing , especially, third-generation cephalosporin (3GC)-resistant strains. To further understand the diversity of 3GC-resistant across animals of different age groups (e.g., pre- and postweaned calves, lactating cows, and dry cows) and farms, we used pulsed-field gel electrophoresis (PFGE) to characterize 70 fecal isolates from 14 dairy farms located in nine Pennsylvania counties. Results of this analysis indicated that 3GC-resistant were highly diverse and grouped into 27 PFGE clades (80% similarity cutoff) and 24 unique antimicrobial resistance patterns were observed among the isolates. For eight farms, clonal with the same resistance patterns were isolated from two or more age groups, indicating that strains were carried in both the calves and adult cows within the same herd. However, there were also several isolates with the same resistance pattern that were distributed to different clades, including isolates from different animal age groups on the same farm, suggesting different strains of within a farm harbored the same resistance-conferring elements. Results of this analysis indicated that 3GC-resistant were highly diverse, associated with multidrug resistance, and circulated through different (noncommingled) animal groups on individual farms.
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http://dx.doi.org/10.1089/fpd.2018.2579DOI Listing
May 2019

Antimicrobial Resistance Among Escherichia coli Isolated from Veal Calf Operations in Pennsylvania.

Foodborne Pathog Dis 2019 01;16(1):74-80

1 Environmental Microbial and Food Safety Laboratory, USDA-Agricultural Research Service, Beltsville, Maryland.

Antimicrobial resistance (AR) is a pressing public health concern, and agricultural operations such as dairy and beef cattle production have been implicated as potential sources of resistant bacteria or genetic elements. This study aimed to determine the prevalence of antimicrobial-resistant Escherichia coli from calf pens in 6 auction houses (56 manure composite samples) and 12 veal calf operations (240 fecal samples in 2 visits: after the calves arrived at the farm and shortly before the animals were sent to slaughter) in the Commonwealth of Pennsylvania. A total of 1567 generic E. coli were isolated and screened for resistance phenotypes. Resistant E. coli were isolated from all auction houses and farms sampled. Based on nonparametric Kruskal-Wallis tests, incremental prevalence of E. coli resistant to ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline in the samples from auction houses and the first and second farm visits was observed (χ 6.98-15.91, p < 0.05). Multidrug-resistant E. coli (resistant to more than three antimicrobial classes) were identified in 76.8%, 90.8%, and 100% of samples collected from the auction houses, first farm visits, and second farm visits, respectively. The presence of bla-E. coli in 11 of the 12 farms presents the possibility of veal production environments being a reservoir for resistant genetic materials that may pose a risk to human health if they are transferred to human pathogens. Additional research on the impact of various management strategies in veal calf rearing is needed for a complete scenario of AR in these production environments.
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http://dx.doi.org/10.1089/fpd.2018.2530DOI Listing
January 2019

Age-Associated Distribution of Antimicrobial-Resistant Salmonella enterica and Escherichia coli Isolated from Dairy Herds in Pennsylvania, 2013-2015.

Foodborne Pathog Dis 2019 01 31;16(1):60-67. Epub 2018 Dec 31.

2 Environmental Microbial and Food Safety Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland.

Antimicrobial resistance has become a major global public health concern, and agricultural operations are often implicated as a source of resistant bacteria. This study characterized the prevalence of antimicrobial-resistant Salmonella enterica and Escherichia coli from a total of 443 manure composite samples from preweaned calves, postweaned calves, dry cows, and lactating cows from 80 dairy operations in Pennsylvania. A total of 1095 S. enterica and 2370 E. coli isolates were screened and tested for resistance to 14 antimicrobials on the National Antimicrobial Resistance Monitoring System Gram-negative (NARMS GN) panel. Salmonellae were isolated from 67% of dairy operations, and 99% of the isolates were pan-susceptible. Salmonella were isolated more frequently from lactating and dry cow samples than from pre- and postweaned calf samples. Overall, the most prevalent serotypes were Cerro, Montevideo, Kentucky, and Newport. E. coli were isolated from all the manure composite samples, and isolates were commonly resistant to tetracyclines, sulfonamides, and aminoglycosides. Resistance was detected more frequently in the E. coli isolates from pre- and postweaned calf samples than in isolates from dry and lactating cow samples (p < 0.05). Multidrug-resistant E. coli (i.e., resistant to >3 antimicrobial classes) were isolated from 66 farms (83%) with significantly greater prevalence in preweaned calves (p < 0.05) than in the older age groups. The bla and bla genes were detected in the cephalosporin-resistant E. coli from 4% and 35% of the farms, respectively. These findings indicate that dairy animals, especially the calf population, serve as significant reservoirs for antimicrobial-resistant bacteria. Additional research on the colonization and persistence of resistant E. coli in calves is warranted to identify potential avenues for mitigation.
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http://dx.doi.org/10.1089/fpd.2018.2519DOI Listing
January 2019

Fecal Metagenome Sequences from Lactating Dairy Cows Shedding Escherichia coli O157:H7.

Microbiol Resour Announc 2018 Nov 8;7(18). Epub 2018 Nov 8.

Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, Agricultural Research Services, United States Department of Agriculture, Beltsville, Maryland, USA.

Cattle are primary reservoirs of Escherichia coli O157:H7, a causative agent of severe human infections. To facilitate analyses of the communities in which this pathogen is found, we sequenced the fecal metagenomes of 10 dairy cows shedding E. coli O157:H7 and added them to the public domain.
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http://dx.doi.org/10.1128/MRA.01279-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256543PMC
November 2018

Genetic diversity and virulence profiles of Listeria monocytogenes recovered from bulk tank milk, milk filters, and milking equipment from dairies in the United States (2002 to 2014).

PLoS One 2018 9;13(5):e0197053. Epub 2018 May 9.

Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, Agricultural Research Services, United States Department of Agriculture, Beltsville, MD, United States of America.

Unpasteurized dairy products are known to occasionally harbor Listeria monocytogenes and have been implicated in recent listeriosis outbreaks and numerous sporadic cases of listeriosis. However, the diversity and virulence profiles of L. monocytogenes isolates recovered from these products have not been fully described. Here we report a genomic analysis of 121 L. monocytogenes isolates recovered from milk, milk filters, and milking equipment collected from bovine dairy farms in 19 states over a 12-year period. In a multi-virulence-locus sequence typing (MVLST) analysis, 59 Virulence Types (VT) were identified, of which 25% were Epidemic Clones I, II, V, VI, VII, VIII, IX, or X, and 31 were novel VT. In a multi-locus sequence typing (MLST) analysis, 60 Sequence Types (ST) of 56 Clonal Complexes (CC) were identified. Within lineage I, CC5 and CC1 were among the most abundant, and within lineage II, CC7 and CC37 were the most abundant. Multiple CCs previously associated with central nervous system and maternal-neonatal infections were identified. A genomic analysis identified variable distribution of virulence markers, Listeria pathogenicity islands (LIPI) -1, -3, and -4, and stress survival island-1 (SSI-1). Of these, 14 virulence markers, including LIPI-3 and -4 were more frequently detected in one lineage (I or II) than the other. LIPI-3 and LIPI-4 were identified in 68% and 28% of lineage I CCs, respectively. Results of this analysis indicate that there is a high level of genetic diversity among the L. monocytogenes present in bulk tank milk in the United States with some strains being more frequently detected than others, and some being similar to those that have been isolated from previous non-dairy related outbreaks. Results of this study also demonstrate significant number of strains isolated from dairy farms encode virulence markers associated with severe human disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197053PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942804PMC
August 2018

Prevalence of Salmonella enterica, Listeria monocytogenes, and pathogenic Escherichia coli in bulk tank milk and milk filters from US dairy operations in the National Animal Health Monitoring System Dairy 2014 study.

J Dairy Sci 2018 Mar 21;101(3):1943-1956. Epub 2017 Dec 21.

Environmental Microbial and Food Safety Laboratory, USDA, Agricultural Research Service, Beltsville, MD 20705. Electronic address:

The dairy farm environment is a well-documented reservoir for zoonotic pathogens such as Salmonella enterica, Shiga-toxigenic Escherichia coli, and Listeria monocytogenes, and humans may be exposed to these pathogens via consumption of unpasteurized milk and dairy products. As part of the National Animal Health Monitoring System Dairy 2014 study, bulk tank milk (BTM, n = 234) and milk filters (n = 254) were collected from a total of 234 dairy operations in 17 major dairy states and analyzed for the presence of these pathogens. The invA gene was detected in samples from 18.5% of operations and Salmonella enterica was isolated from 18.0% of operations. Salmonella Dublin was detected in 0.7% of operations. Sixteen Salmonella serotypes were isolated, and the most common serotypes were Cerro, Montevideo, and Newport. Representative Salmonella isolates (n = 137) were tested against a panel of 14 antimicrobials. Most (85%) were pansusceptible; the remaining were resistant to 1 to 9 antimicrobials, and within the resistant strains the most common profile was resistance to ampicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Listeria spp. were isolated from 19.9% of operations, and L. monocytogenes was isolated from 3.0% of operations. Serogroups 1/2a and 1/2b were the most common, followed by 4b and 4a. One or more E. coli virulence genes were detected in the BTM from 30.5% of operations and in the filters from 75.3% of operations. A combination of stx, eaeA, and γ-tir genes was detected in the BTM from 0.5% of operations and in the filters from 6.6% of operations. The results of this study indicate an appreciable prevalence of bacterial pathogens in BTM and filters, including serovars known to infect humans.
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http://dx.doi.org/10.3168/jds.2017-13546DOI Listing
March 2018

Genome Sequences of subsp. Serovar Kentucky Sequence Type 152 Isolated from Dairy Cows in the United States.

Genome Announc 2017 Oct 19;5(42). Epub 2017 Oct 19.

Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, USDA-ARS, Beltsville, Maryland, USA

subsp. serovar Kentucky ( Kentucky) is frequently isolated from dairy cows in the United States, but is an infrequent cause of human salmonellosis. To investigate the genomic features of  Kentucky strains isolated from a single dairy farm, the genomes of eight isolates were sequenced and added to the public domain.
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http://dx.doi.org/10.1128/genomeA.01119-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646404PMC
October 2017

Genome Sequences of 30 O157:H7 Isolates Recovered from a Single Dairy Farm and Its Associated Off-Site Heifer-Raising Facility.

Genome Announc 2017 Aug 31;5(35). Epub 2017 Aug 31.

Environmental Microbial and Food Safety Laboratory, Beltsville Area Research Center, Agricultural Research Services, United States Department of Agriculture, Beltsville, Maryland, USA

Cattle are the primary reservoir of O157:H7, the most frequently isolated serotype of enterohemorrhagic infections among humans in North America. To evaluate the diversity of O157:H7 isolates within a single dairy herd, the genomes of 30 isolates collected over a 7-year period were sequenced.
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http://dx.doi.org/10.1128/genomeA.00814-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578836PMC
August 2017

Genome Sequences of Five Multidrug-Resistant Sequence Type 117 Isolates Recovered from Dairy Calves.

Genome Announc 2017 Aug 17;5(33). Epub 2017 Aug 17.

Environmental Microbial and Food Safety Laboratory, Beltsville Area Research Center, Agricultural Research Services, United States Department of Agriculture, Beltsville, Maryland, USA

sequence type 117 (ST117) strains have been recovered from poultry with colibacillosis, as well as from urinary tract infections and fatal septic infections in humans. To further investigate ST117 isolates recovered from nonpoultry food animals, we sequenced the genomes of five ST117 isolates from dairy calves in Pennsylvania.
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http://dx.doi.org/10.1128/genomeA.00732-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604762PMC
August 2017

Prevalence, antimicrobial resistance, and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies.

J Dairy Sci 2017 May 23;100(5):3470-3479. Epub 2017 Feb 23.

Environmental Microbial and Food Safety Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705. Electronic address:

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100-499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30-99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.
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http://dx.doi.org/10.3168/jds.2016-12084DOI Listing
May 2017

Genomic and Evolutionary Analysis of Two Salmonella enterica Serovar Kentucky Sequence Types Isolated from Bovine and Poultry Sources in North America.

PLoS One 2016 3;11(10):e0161225. Epub 2016 Oct 3.

Environmental Microbial and Food Safety Laboratory, Beltsville Area Research Center, Agricultural Research Services, United States Department of Agriculture, Beltsville, MD, United States of America.

Salmonella enterica subsp. enterica serovar Kentucky is frequently isolated from healthy poultry and dairy cows and is occasionally isolated from people with clinical disease. A genomic analysis of 119 isolates collected in the United States from dairy cows, ground beef, poultry and poultry products, and human clinical cases was conducted. Results of the analysis demonstrated that the majority of poultry and bovine-associated S. Kentucky were sequence type (ST) 152. Several bovine-associated (n = 3) and food product isolates (n = 3) collected from the United States and the majority of human clinical isolates were ST198, a sequence type that is frequently isolated from poultry and occasionally from human clinical cases in Northern Africa, Europe and Southeast Asia. A phylogenetic analysis indicated that both STs are more closely related to other Salmonella serovars than they are to each other. Additionally, there was strong evidence of an evolutionary divergence between the poultry-associated and bovine-associated ST152 isolates that was due to polymorphisms in four core genome genes. The ST198 isolates recovered from dairy farms in the United States were phylogenetically distinct from those collected from human clinical cases with 66 core genome SNPs differentiating the two groups, but more isolates are needed to determine the significance of this distinction. Identification of S. Kentucky ST198 from dairy animals in the United States suggests that the presence of this pathogen should be monitored in food-producing animals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161225PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047448PMC
June 2017

Comparison of Microbial Communities Isolated from Feces of Asymptomatic Salmonella-Shedding and Non-Salmonella Shedding Dairy Cows.

Front Microbiol 2016 1;7:691. Epub 2016 Jun 1.

Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Agricultural Research Service Beltsville, MD, USA.

In the United States Salmonella enterica subsp. enterica serotypes Kentucky and Cerro are frequently isolated from asymptomatic dairy cows. However, factors that contribute to colonization of the bovine gut by these two serotypes have not been identified. To investigate associations between Salmonella status and bacterial diversity, as well as the diversity of the microbial community in the dairy cow hindgut, the bacterial and archaeal communities of fecal samples from cows on a single dairy farm were determined by high-throughput sequencing of 16S rRNA gene amplicons. Fecal grab samples were collected from two Salmonella-positive cows and two Salmonella-negative cows on five sampling dates (n = 20 cows), and 16S rRNA gene amplicons from these samples were sequenced on the Illumina MiSeq platform. A high level of alpha (within) and beta diversity (between) samples demonstrated that microbial profiles of dairy cow hindguts are quite diverse. To determine whether Salmonella presence, sampling year, or sampling date explained a significant amount of the variation in microbial diversity, we performed constrained ordination analyses (distance based RDA) on the unifrac distance matrix produced with QIIME. Results indicated that there was not a significant difference in the microbial diversity associated with Salmonella presence (P > 0.05), but there were significant differences between sampling dates and years (Pseudo-F = 2.157 to 4.385, P < 0.05). Based on these data, it appears that commensal Salmonella infections with serotypes Cerro and Kentucky in dairy cows have little or no association with changes in the abundance of major bacterial groups in the hindgut. Rather, our results indicated that temporal dynamics and other undescribed parameters associated with them were the most influential drivers of the differences in microbial diversity and community structure in the dairy cow hindgut.
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http://dx.doi.org/10.3389/fmicb.2016.00691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887466PMC
June 2016

Complete Genome Sequence and Methylome of Salmonella enterica subsp. enterica Cerro, a Frequent Dairy Cow Serovar.

Genome Announc 2016 Jan 28;4(1). Epub 2016 Jan 28.

Beltsville Agricultural Research Center, Environmental Microbial and Food Safety Laboratory, USDA-ARS, Beltsville, Maryland, USA

Salmonella enterica subsp. enterica serovar Cerro is an infrequent pathogen of humans and other mammals but is frequently isolated from the hindgut of asymptomatic cattle in the United States. To further understand the genomic determinants of S. Cerro specificity for the bovine hindgut, the genome of isolate CFSAN001588 was fully sequenced and deposited in the GenBank database.
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http://dx.doi.org/10.1128/genomeA.01350-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732324PMC
January 2016

Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria.

Environ Monit Assess 2016 Jan 24;188(1):56. Epub 2015 Dec 24.

USDA-ARS Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, 10300 Baltimore Ave. Bldg. 173, Beltsville, MD, 20705, USA.

The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hot spot for proliferation and gene exchange. Pipe-based irrigation systems that utilize surface waters may contribute to the dissemination of antibiotic-resistant bacteria in a similar manner. We conducted irrigation events at a perennial stream on a weekly basis for 1 month, and the concentrations of total heterotrophic bacteria, total coliforms, and fecal coliforms, as well as the concentrations of these bacterial groups that were resistant to ampicillin and tetracycline, were monitored at the intake water. Prior to each of the latter three events, residual pipe water was sampled and 6-in. sections of pipeline (coupons) were detached from the system, and biofilm from the inner-wall was removed and analyzed for total protein content and the above bacteria. Isolates of biofilm-associated bacteria were screened for resistance to a panel of seven antibiotics, representing five antibiotic classes. All of the monitored bacteria grew substantially in the residual water between irrigation events, and the biomass of the biofilm steadily increased from week to week. The percentages of biofilm-associated isolates that were resistant to antibiotics on the panel sometimes increased between events. Multiple-drug resistance was observed for all bacterial groups, most often for fecal coliforms, and the distributions of the numbers of antibiotics that the total coliforms and fecal coliforms were resistant to were subject to change from week to week. Results from this study highlight irrigation waters as a potential source for antibiotic-resistant bacteria, which can subsequently become incorporated into and proliferate within irrigation pipe-based biofilms.
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http://dx.doi.org/10.1007/s10661-015-5067-4DOI Listing
January 2016

Diversity of Listeria monocytogenes within a U.S. dairy herd, 2004-2010.

Foodborne Pathog Dis 2015 Oct 1;12(10):844-50. Epub 2015 Sep 1.

1 Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center , Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland.

Listeria monocytogenes, the causative agent of listeriosis, is frequently isolated from the environment. Dairy cows and dairy farm environments are reservoirs of this pathogen, where fecal shedding contributes to its environmental dispersal and contamination of milk, dairy products, and meat. The molecular diversity of 40 L. monocytogenes isolates representing 3 serogroups (1/2a, 1/2b, and 4b) collected between 2004 and 2010 from the feces of dairy cattle on a single dairy farm was assessed using a multivirulence locus sequence typing (MVLST) assay. The dairy farm L. monocytogenes MVLST patterns were compared to those from 138 strains isolated globally from clinical cases, foods, and the environment. Results of the study demonstrated that several distantly related L. monocytogenes strains persisted among members of the herd over the course of the study while other strains were transient. Furthermore, some strains isolated during this study appear to be distantly related to previously isolated L. monocytogenes while others are closely related to Epidemic Clones associated with human illness. This work demonstrates that dairy cows can be reservoirs of a diverse population of potentially human pathogenic L. monocytogenes that represents a risk to consumers of milk, dairy products, and meat.
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http://dx.doi.org/10.1089/fpd.2014.1886DOI Listing
October 2015

Improvements to a PCR-Based Serogrouping Scheme for Salmonella enterica from Dairy Farm Samples.

J Food Prot 2015 Jun;78(6):1182-5

U.S. Department of Agriculture, Agricultural Research Service, Henry A. Wallace Agricultural Research Center, Environmental Microbial and Food Safety Laboratory, Beltsville, Maryland 20705, USA.

Molecular serotyping through the use of PCR is a simple and useful technique for characterizing isolates of Salmonella enterica subsp. enterica belonging to serogroups B, C1, C2, D1, and E1, which are the majority of the isolates associated with human disease outbreaks. However, many of the Salmonella strains currently isolated from dairy farms in the northeastern United States are serovar Cerro, a group K strain not detected by this assay. Primers from a well-known PCR assay for the identification of Salmonella were added to a commonly used serotyping assay so that strains, such as Salmonella Cerro, that do not produce bands in the original assay can be confirmed as belonging to S. enterica subsp. enterica. The modified assay frequently misidentified the serogroup of Salmonella Mbandaka isolates because of failure to amplify the wzxC1 amplicon. Therefore, the reverse primer for the wzxC1 target was modified based on in silico analysis to provide consistent classification of Salmonella Mbandaka as belonging to serogroup C1. These two modifications to the serogrouping PCR method enhance the utility of the method for characterizing Salmonella isolates.
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http://dx.doi.org/10.4315/0362-028X.JFP-14-475DOI Listing
June 2015

Dynamics of Escherichia coli Virulence Factors in Dairy Herds and Farm Environments in a Longitudinal Study in the United States.

Appl Environ Microbiol 2015 Jul 24;81(13):4477-88. Epub 2015 Apr 24.

Department of Nutrition and Food Science, University of Maryland, College Park, Maryland, USA Center for Food Safety and Security Systems, University of Maryland, College Park, Maryland, USA

Pathogenic Escherichia coli or its associated virulence factors have been frequently detected in dairy cow manure, milk, and dairy farm environments. However, it is unclear what the long-term dynamics of E. coli virulence factors are and which farm compartments act as reservoirs. This study assessed the occurrence and dynamics of four E. coli virulence factors (eae, stx1, stx2, and the gamma allele of the tir gene [γ-tir]) on three U.S. dairy farms. Fecal, manure, water, feed, milk, and milk filter samples were collected from 2004 to 2012. Virulence factors were measured by postenrichment quantitative PCR (qPCR). All factors were detected in most compartments on all farms. Fecal and manure samples showed the highest prevalence, up to 53% for stx and 21% for γ-tir in fecal samples and up to 84% for stx and 44% for γ-tir in manure. Prevalence was low in milk (up to 1.9% for stx and 0.7% for γ-tir). However, 35% of milk filters were positive for stx and 20% were positive for γ-tir. All factors were detected in feed and water. Factor prevalence and levels, expressed as qPCR cycle threshold categories, fluctuated significantly over time, with no clear seasonal signal independent from year-to-year variability. Levels were correlated between fecal and manure samples, and in some cases autocorrelated, but not between manure and milk filters. Shiga toxins were nearly ubiquitous, and 10 to 18% of the lactating cows were potential shedders of E. coli O157 at least once during their time in the herds. E. coli virulence factors appear to persist in many areas of the farms and therefore contribute to transmission dynamics.
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http://dx.doi.org/10.1128/AEM.00465-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475889PMC
July 2015

Genome Sequences of Eight Salmonella enterica subsp. enterica Serovars Isolated from a Single Dairy Farm.

Genome Announc 2014 Apr 24;2(2). Epub 2014 Apr 24.

Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, U.S. Department of Agriculture, ARS, Beltsville, Maryland, USA.

Here, we report draft genome sequences of 26 isolates of Salmonella enterica subsp. enterica, representing eight serotypes, which were isolated from cows in a Pennsylvania dairy herd, the farm on which they were reared, and the associated off-site heifer-raising facility over an 8-year sampling period.
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http://dx.doi.org/10.1128/genomeA.00082-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999488PMC
April 2014

Stochastic modeling of imperfect Salmonella vaccines in an adult dairy herd.

Bull Math Biol 2014 Mar 28;76(3):541-65. Epub 2014 Feb 28.

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA,

Salmonella is a major cause of bacterial foodborne disease. Human salmonellosis results in significant public health concerns and a considerable economic burden. Dairy cattle are recognized as a key source of several Salmonella serovars that are a threat to human health. To lower the risk of Salmonella infection, reduction of Salmonella prevalence in dairy cattle is important. Vaccination as a control measure has been applied for reduction of preharvest Salmonella prevalence on dairy farms. Salmonella vaccines are usually imperfect (i.e., vaccines may provide a partial protection for susceptible animals, reduce the infectiousness and shedding level, shorten the infectious period of infected animals, and/or curb the number of clinical cases), and evaluation of the potential impacts of imperfect Salmonella vaccines at the farm level is valuable to design effective intervention strategies. The objective of this study was to investigate the impact of imperfect Salmonella vaccines on the stochastic transmission dynamics in an adult dairy herd. To this end, we developed a semi-stochastic and individual-based continuous time Markov chain (CTMC) vaccination model with both direct and indirect transmission, and applied the CTMC vaccination model to Salmonella Cerro transmission in an adult dairy herd. Our results show that vaccines shortening the infectious period are most effective in reducing prevalence, and vaccines decreasing host susceptibility are most effective in reducing the outbreak size. Vaccines with multiple moderate efficacies may have the same effectiveness as vaccines with a single high efficacy in reducing prevalence, time to extinction, and outbreak size. Although the environment component has negligible contributions to the prevalence, time to extinction, and outbreak size for Salmonella Cerro in the herd, the relative importance of environment component was not assessed. This study indicates that an effective vaccination program against Salmonella Cerro spread in the herd can be designed with (1) vaccines with a single high efficacy in reducing either the infectious period or susceptibility of the host, or (2) if such single high efficacy vaccines are not available, vaccines with multiple moderate efficacies may be considered instead. These findings are also of general value for designing vaccination program for Salmonella serotypes in livestock.
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http://dx.doi.org/10.1007/s11538-013-9931-5DOI Listing
March 2014

Invasion and transmission of Salmonella Kentucky in an adult dairy herd using approximate Bayesian computation.

BMC Vet Res 2013 Dec 5;9:245. Epub 2013 Dec 5.

Section of Epidemiology, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Background: An outbreak of Salmonella Kentucky followed by a high level of sustained endemic prevalence was recently observed in a US adult dairy herd enrolled in a longitudinal study involving intensive fecal sampling. To understand the invasion ability and transmission dynamics of Salmonella Kentucky in dairy cattle, accurate estimation of the key epidemiological parameters from longitudinal field data is necessary. The approximate Bayesian computation technique was applied for estimating the transmission rate (β), the recovery rate (γ) and shape (n) parameters of the gamma distribution for the infectious (shedding) period, and the basic reproduction ratio (R0), given a susceptible-infectious-recovered-susceptible (SIRS) compartment model with a gamma distribution for the infectious period.

Results: The results report that the mean transmission rate (β) is 0.417 month-1 (median: 0.417, 95% credible interval [0.406, 0.429]), the average infectious period (γ-1) is 7.95 months (median: 7.95, 95% credible interval [7.70, 8.22]), the mean shape parameter (n) of the gamma distribution for the infectious period is 242 (median: 182, 95% credible interval [16, 482]), and the mean basic reproduction ratio (R0) is 2.91 (median: 2.91, 95% credible interval [2.83, 3.00]).

Conclusions: This study shows that Salmonella Kentucky in this herd was of mild infectiousness and had a long infectious period, which together provide an explanation for the observed prevalence pattern after invasion. The transmission rate and the recovery rate parameters are inferred with better accuracy than the shape parameter, therefore these two parameters are more sensitive to the model and the observed data. The estimated shape parameter (n) has large variability with a minimal value greater than one, indicating that the infectious period of Salmonella Kentucky in dairy cattle does not follow the conventionally assumed exponential distribution.
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http://dx.doi.org/10.1186/1746-6148-9-245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4235045PMC
December 2013

Phylogenetic diversity of the enteric pathogen Salmonella enterica subsp. enterica inferred from genome-wide reference-free SNP characters.

Genome Biol Evol 2013 ;5(11):2109-23

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD.

The enteric pathogen Salmonella enterica is one of the leading causes of foodborne illness in the world. The species is extremely diverse, containing more than 2,500 named serovars that are designated for their unique antigen characters and pathogenicity profiles-some are known to be virulent pathogens, while others are not. Questions regarding the evolution of pathogenicity, significance of antigen characters, diversity of clustered regularly interspaced short palindromic repeat (CRISPR) loci, among others, will remain elusive until a strong evolutionary framework is established. We present the first large-scale S. enterica subsp. enterica phylogeny inferred from a new reference-free k-mer approach of gathering single nucleotide polymorphisms (SNPs) from whole genomes. The phylogeny of 156 isolates representing 78 serovars (102 were newly sequenced) reveals two major lineages, each with many strongly supported sublineages. One of these lineages is the S. Typhi group; well nested within the phylogeny. Lineage-through-time analyses suggest there have been two instances of accelerated rates of diversification within the subspecies. We also found that antigen characters and CRISPR loci reveal different evolutionary patterns than that of the phylogeny, suggesting that a horizontal gene transfer or possibly a shared environmental acquisition might have influenced the present character distribution. Our study also shows the ability to extract reference-free SNPs from a large set of genomes and then to use these SNPs for phylogenetic reconstruction. This automated, annotation-free approach is an important step forward for bacterial disease tracking and in efficiently elucidating the evolutionary history of highly clonal organisms.
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http://dx.doi.org/10.1093/gbe/evt159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3845640PMC
October 2014

Regional distribution of two dairy-associated Salmonella enterica serotypes.

Foodborne Pathog Dis 2013 May 28;10(5):448-52. Epub 2013 Mar 28.

Environmental Microbial and Food Safety Laboratory, Beltsville Agricultual Research Center, U.S. Department of Agriculture-Agricultural Research Service, Beltsville, Maryland 20705, USA.

Salmonella enterica is a zoonotic pathogen that is often associated with dairy farms. The organism can cause disease in cows but is also frequently shed in large numbers by dairy cows that are asymptomatic. Long-term asymptomatic infections with serotypes Cerro and Kentucky were previously identified in cows on a 100-head dairy farm in Pennsylvania, United States (focal dairy). Milk filters were collected from farms within 30 miles of the focal dairy to determine whether the infections by Cerro and Kentucky were limited to the focal dairy or whether the infection might be more regional in nature. Analysis of milk filters showed that Cerro and Kentucky were widespread in the surrounding region with 16 of 39 farms (41%) positive for one or both serotypes. Pulsed-field gel electrophoresis showed that the milk filter Kentucky strains shared >90% similarity with strains from the focal dairy and from local streams. Although there was more variation between Cerro strains (>80% similarity), most milk filter Cerro isolates from most milk filters were highly similar (>90%) to strains isolated from the focal dairy and local streams. In this intensely dairy-farmed region, Salmonella infection of dairy cows appears to be regional in nature, a fact that will impact efforts to control these pathogens.
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http://dx.doi.org/10.1089/fpd.2012.1380DOI Listing
May 2013

Effects of temperature on bacterial transport and destruction in bioretention media: field and laboratory evaluations.

Water Environ Res 2012 Jun;84(6):485-96

Department of Civil and Environmental Engineering, University of Maryland, College Park, MD, USA.

Microbial activities are significantly influenced by temperature. This study investigated the effects of temperature on the capture and destruction of bacteria from urban stormwater runoff in bioretention media using 2-year field evaluations coupled with controlled laboratory column studies. Field data from two bioretention cells show that the concentration of indicator bacteria (fecal coliforms and Escherichia coli) was reduced during most storm events, and that the probability of meeting specific water quality criteria in the discharge was increased. Indicator bacteria concentration in the input flow typically increased with higher daily temperature. Although bacterial removal efficiency was independent of temperature in the field and laboratory, column tests showed that bacterial decay coefficients in conventional bioretention media (CBM) increase exponentially with elevated temperature. Increases in levels of protozoa and heterotrophic bacteria associated with increasing temperature appear to contribute to faster die-off of trapped E. coli in CBM via predation and competition.
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http://dx.doi.org/10.2175/106143012x13280358613589DOI Listing
June 2012

Dynamics of Salmonella serotype shifts in an endemically infected dairy herd.

Foodborne Pathog Dis 2012 Apr 2;9(4):319-24. Epub 2012 Feb 2.

Environmental Microbial and Food Safety Laboratory, ANRI, U.S. Department of Agriculture-Agricultural Research Service, Beltsville, MD 20705, USA.

Salmonella is a leading cause of foodborne illness in the United States. It is a zoonotic pathogen found in many species of food animals, and contamination of foodstuffs by strains of Salmonella found on farms is an important source of human exposure. Here we describe a long-term (2004-2010) study of Salmonella colonization on a typical dairy farm in the Northeastern United States. The fecal shedding prevalence in the herd ranged from 8% to 97%, and greater than 50% of the herd was shedding Salmonella for more than two-thirds of the study period. Salmonella enterica serotype Cerro was first detected in September 2004, after a small and very short-lived outbreak of Salmonella Kentucky. Cerro persisted within the herd for over 3 years, with no clinical signs of salmonellosis in the animals. In the winter of 2006, Kentucky was again detected within the herd, and over a 2-year period, Kentucky gradually supplanted Cerro. Kentucky was the only serotype detected from March 2008 until September 2009, when Cerro was again detected in 15% of the cows on the farm. Since September 2009, Kentucky and Cerro have coexisted within the herd, which continues to harbor these serotypes at high prevalence. Pulsed-field gel electrophoresis (PFGE) could not discern differences between Cerro strains isolated during this study, but it did suggest that the strain of Kentucky that seemed to behave as a commensal in these dairy cows is distinct from the transient strain isolated in 2004. Understanding the dynamics of competition between these two serotypes that seem to behave as commensal colonizers of dairy cows may provide insights into the mechanisms by which Salmonella establishes infection in the lower gut of dairy cows and may lead to the development of measures to prevent or limit Salmonella colonization of dairy cows.
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http://dx.doi.org/10.1089/fpd.2011.1054DOI Listing
April 2012

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay.

Biosens Bioelectron 2011 Dec 1;30(1):337-41. Epub 2011 Oct 1.

Creatv MicroTech, Inc., Potomac, MD 20854, USA.

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10(5) cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10-10(4) cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.
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http://dx.doi.org/10.1016/j.bios.2011.09.029DOI Listing
December 2011

Quantitative risk assessment of listeriosis due to consumption of raw milk.

J Food Prot 2011 Aug;74(8):1268-81

Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, New York 14850, USA.

The objectives of this study were to estimate the risk of illness for raw milk consumers due to Listeria monocytogenes in raw milk sold by permitted dealers, and the risk for people on farms who consume raw milk. Three scenarios were evaluated for raw milk sold by dealers: raw milk purchased directly from bulk tanks, from on-farm stores, and from retail. To assess the effect of mandatory testing of raw milk by regulatory agencies, the number of listeriosis cases per year was compared where no raw milk testing was done, only a screening test to issue a permit was conducted, and routine testing was conducted and milk was recalled if it was L. monocytogenes positive. The median number of listeriosis cases associated with consumption of raw milk from bulk tanks, farm stores, and retail for an intermediate-age population was 6.6 × 10(-7), 3.8 × 10(-5), and 5.1 × 10(-5) cases per year, respective ly. In populations with high susceptibility, the estimated median number of cases per year was 2.7 × 10(-7) (perinatal, i.e., pregnant women and their fetuses or newborns) and 1.4 × 10(-6) (elderly) for milk purchased from bulk tanks, 1.5 × 10(-5 ) (perinatal) and 7.8 × 10(-5) (elderly) for milk from farm stores, and 2.1 × 10(-5) (perinatal) and 1.0 × 10(-4) (elderly) for milk from retail. For raw milk consumed on farms, the median number of listeriosis cases was 1.4 × 10(-7) cases per year. A greater risk of listeriosis was associated with consumption of raw milk obtained from retail and farm stores as compared with milk obtained from bulk tanks. This was likely due to additional time-temperature combination steps in the retail and farm store models, which increased the chances for growth of L. monocytogenes in raw milk. A close relationship between prevalence of L. monocytogenes in raw milk and the values of disease incidence was observed. Hence, a reduction in the number of cases per year in all populations was observed when a raw milk-testing program was in place, especially when routine testing and recalling of milk was conducted.
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http://dx.doi.org/10.4315/0362-028X.JFP-10-554DOI Listing
August 2011

Prevalence of Salmonella enterica, Listeria monocytogenes, and Escherichia coli virulence factors in bulk tank milk and in-line filters from U.S. dairies.

J Food Prot 2011 May;74(5):759-68

Environmental Microbial and Food Safety Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Building 173, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350, USA.

The zoonotic bacteria Salmonella enterica, Listeria monocytogenes, and Escherichia coli are known to infect dairy cows while not always causing clinical signs of disease. These pathogens are sometimes found in raw milk, and human disease outbreaks due to these organisms have been associated with the consumption of raw milk or raw milk products. Bulk tank milk (BTM) samples (536) and in-line milk filters (519) collected from dairy farms across the United States during the National Animal Health Monitoring System's Dairy 2007 study were analyzed by real-time PCR for the presence of S. enterica and pathogenic forms of E. coli and by culture techniques for the presence of L. monocytogenes. S. enterica was detected in samples from 28.1% of the dairy operations, primarily in milk filters. Salmonella was isolated from 36 of 75 PCR-positive BTM samples and 105 of 174 PCR-positive filter samples, and the isolates were serotyped. Cerro, Kentucky, Muenster, Anatum, and Newport were the most common serotypes. L. monocytogenes was isolated from 7.1% of the dairy operations, and the 1/2a complex was the most common serotype, followed by 1/2b and 4b (lineage 1). Shiga toxin genes were detected in enrichments from 15.2% of the BTM samples and from 51.0% of the filters by real-time PCR. In most cases, the cycle threshold values for the PCR indicated that toxigenic strains were not a major part of the enrichment populations. These data confirm those from earlier studies showing significant contamination of BTM by zoonotic bacterial pathogens and that the consumption of raw milk and raw milk products presents a health risk.
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http://dx.doi.org/10.4315/0362-028X.JFP-10-423DOI Listing
May 2011

Increased in vitro adherence and on-farm persistence of predominant and persistent Listeria monocytogenes strains in the milking system.

Appl Environ Microbiol 2011 Jun 25;77(11):3676-84. Epub 2011 Mar 25.

Department of Pathology and Preventive Medicine, College of Veterinary Sciences-Universidad de Concepcion, Av. Vincente Mendez no. 595, Chillan, Chile.

Dairy farms are a reservoir for Listeria monocytogenes, and the reduction of this pathogen at the farm level is important for reducing human exposure. The objectives of this research were to study the diversity of L. monocytogenes strains on a single dairy farm, assess strain dynamics within the farm, identify potential sources of L. monocytogenes in bulk tank milk and milk filters, and assess the adherence abilities of representative strains. A total of 248 L. monocytogenes isolates were analyzed by pulsed-field gel electrophoresis (PFGE). Combined AscI and ApaI restriction analysis yielded 40 PFGE types (strains). The most predominant strains were T (28.6%), D (22.6%), and F (14.9%). A high level of heterogeneity of strains among isolates from fecal (Simpson's index of diversity [SID] = 0.96) and environmental (SID = 0.96) samples was observed. A higher homogeneity of strains was observed among isolates from milk filters (SID = 0.71) and bulk tank milk (SID = 0.65). Six of 17 L. monocytogenes isolates (35.3%) were classified in an in vitro assay as having a "low adherence ability," 9 (52.9%) were classified as having a "medium adherence ability," and 2 (11.8%) were classified as having a "high adherence ability." The L. monocytogenes strains that were predominant and persistent showed significantly better adherence than did strains that were only sporadic, predominant, or persistent (P = 0.0006). Our results suggest that the milking system was exposed to several L. monocytogenes strains from different sources. Only 3 strains, however, were successful in persisting within the milking system, suggesting that some strains are more suitable to that particular ecological environment than others.
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http://dx.doi.org/10.1128/AEM.02441-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127605PMC
June 2011

Relationship between eae and stx virulence genes and Escherichia coli in an agricultural watershed: implications for irrigation water standards and leafy green commodities.

J Food Prot 2011 Jan;74(1):18-23

U.S. Department of Agriculture, Agricultural Research Service, Environmental Microbial and Food Safety Laboratory, Building 173, Beltsville Agricultural Research Center-East, 10300 Baltimore Avenue, Beltsville, Maryland 20705, USA.

The California Leafy Greens Marketing Agreement (LGMA) was adopted in an effort to minimize the risk of contamination of leafy greens with enteric pathogens from a variety of sources, including ground and surface irrigation waters. The LGMA contains standards similar to those established for recreational waters, based on Escherichia coli concentrations. However, no correlation between E. coli and any specific waterborne pathogen(s) has been reported. We conducted this monitoring study in an agricultural watershed to (i) evaluate spatial and temporal fluctuations in E. coli populations and virulence genes associated with pathogenic E. coli and (ii) investigate whether a relationship could be established between E. coli and virulence genes. The virulence genes targeted for analysis were the eae and stx genes, encoding for intimin and Shiga-like toxins, respectively; they were detected with PCR methods. E. coli concentrations and eae and stx prevalence varied both spatially and temporally. In general, both were higher in agricultural than in forested areas and were higher in the summer and fall seasons than in winter. The eae and stx genes were prevalent throughout the watershed. However, in the absence of actual isolates, no conclusions could be drawn regarding the prevalence of specific pathogenic E. coli. No correlation was observed between E. coli concentrations and virulence genes; lower E. coli concentrations were not necessarily associated with decreased prevalence of eae and stx genes. These results suggest that the LGMA standards might not adequately address the issue of waterborne contamination, and that alternative criteria might be required.
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http://dx.doi.org/10.4315/0362-028X.JFP-10-241DOI Listing
January 2011

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis in a longitudinal study of three dairy herds.

J Clin Microbiol 2011 Mar 5;49(3):893-901. Epub 2011 Jan 5.

Quality Milk Production Services, Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, New York 14853, USA.

The objective of this study was to evaluate whether cows that were low shedders of Mycobacterium avium subsp. paratuberculosis were passively shedding or truly infected with M. avium subsp. paratuberculosis. We also investigated whether it is possible that these M. avium subsp. paratuberculosis-infected animals could have been infected as adults by contemporary high-shedding animals (supershedders). The M. avium subsp. paratuberculosis isolates were obtained from a longitudinal study of three dairy herds in the northeastern United States. Isolates were selected from fecal samples and tissues at slaughter from all animals that were culture positive at the same time that supershedders were present in the herds. Shedding levels (CFU of M. avium subsp. paratuberculosis/g of feces) for the animals at each culture-positive occasion were determined. Using a multilocus short-sequence-repeat technique, we found 15 different strains of M. avium subsp. paratuberculosis from a total of 142 isolates analyzed. Results indicated herd-specific infection patterns; there was a clonal infection in herd C, with 89% of isolates from animals sharing the same strain, whereas herds A and B showed several different strains infecting the animals at the same time. Tissues from 80% of cows with at least one positive fecal culture (other than supershedders) were culture positive, indicating a true M. avium subsp. paratuberculosis infection. The results of M. avium subsp. paratuberculosis strain typing and observed shedding levels showed that at least 50% of low shedders have the same strain as that of a contemporary supershedder. Results of this study suggest that in a dairy herd, more of the low-shedding cows are truly infected with M. avium subsp. paratuberculosis than are passively shedding M. avium subsp. paratuberculosis. The sharing of strains between low shedders and the contemporary supershedders suggests that low shedders may have been infected by environmental exposure of M. avium subsp. paratuberculosis.
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http://dx.doi.org/10.1128/JCM.01107-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067694PMC
March 2011
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